The invention discloses an innovative method and apparatus by which bubbles may be suppressed in optical measurement cells, including, but not limited to electrophoretic mobility measurement cells, wherein the motion of charged particles in a solution subject to an applied electric field is measured. Although the present invention will refer to macromolecules throughout much of its specification, measurement cells capable of bubble suppression using the inventive method and apparatus disclosed herein may include more generally all classes of small particles including emulsions, viruses, nanoparticles, liposomes, macro-ions and any other solution constituents whose size may lie between about a half and a few thousand nanometers. Thus whenever the terms “molecule,” “macromolecule,” or “macro-ion” are used, it should be understood to include all of the aforementioned solution-borne objects to be subject to some form of optical measurement.
Electrophoretic mobility is the directly measurable and most widely used quantity to characterize the charge of molecules, or other particles in solution. Once measured, the electrophoretic mobility can be used in turn to determine the effective charge, Ze, carried by such molecules as well as their so-called zeta potential ζ. The interface between the group of ions tightly bound to the particle and those of the surrounding solution that do not move with the particle defines the hydrodynamic shear plane. The zeta potential represents the electrostatic potential existing at this shear plane. It is an objective of the present invention to improve optical measurements of electrophoretic mobility, effective charge, and zeta potential of molecules and particles in solution contained within an optical measurement cell. It should be noted, however, that the present inventive method and apparatus is not limited to measurements based on particle charge. Other measurements, such as multi-angle light scattering, MALS, and quasi-elastic light scattering, QELS, also often referred to as dynamic light scattering, DLS, wherein a light source is passed through an optically transparent measurement cell to illuminate a liquid sample contained therein, are among the other techniques which will benefit from the inventive elements disclosed herein. Therefore, while this disclosure will focus mainly on the utility of the invention as applied to mobility measurement cells, it should in no way be considered limited to this measurement technique or application.
Several techniques have been developed and are available for measuring mobilities including light scattering methods such as heterodyne DLS including both laser Doppler electrophoresis, LDE, and phase analysis light scattering, PALS. These techniques involve measuring light scattered from moving particles, whereby such scattered light carries information relating to such motion and from which the associated electrophoretic mobility of the particles may be determined.
The most significant of these techniques for the measurement of electrophoretic mobilities is PALS, where a beam of monochromatic light, usually from a laser source, illuminates a sample of liquid borne particles exposed to an applied electric field. Some of the light they scatter is collected and combined with a fraction of the incident, unscattered light. In other words, the scattered signal is combined coherently with the incident light to produce a heterodyned signal producing interference fringes at a detector. In order to measure the fluctuations of the combined beams and derive therefrom measurement of the electrophoretic mobility of the scattering particles, the incident beam fraction is directed to reflect from an oscillating mirror. This causes the detected fringes to exhibit an intensity modulation, even in the absence of electrophoretic motion. The electrophoretic motion that results from the application the applied field produces an additive frequency shift permitting, thereby, an unequivocal determination of the direction of the particles relative to the direction of the applied electric field. This process requires very precise measurement of light scattered from the sample. Bubbles contained within the illuminated sample scatter light that interferes with that scattered from the molecules, corrupting the derived measurements.
When making light scattering measurements, maintaining a bubble free environment is always a challenge. There are many ways of attempting to introduce a bubble free sample to a measurement cell including, but not limited to: vibrating, tapping, rinsing with alcohol, or even intentionally flushing the cell with air. While these may be helpful, it often happens that bubbles adhere to the surfaces. Even if a bubble-free sample is achieved, the electrical current that gives rise to the electrophoretic molecular motion can also cause electrolysis of the solvent resulting in bubbles that form on the electrodes. The problem becomes worse as the ionic strength of the buffer is increased because a progressively larger current is required to achieve a given electric field. Large currents generate more bubbles. Another objective of the present invention is to reduce the number and size of such bubbles that can interfere with electrophoretic measurements.
Additionally, samples that contain gas in solution or that undergo a chemical reaction, may spontaneously generate bubbles even in the absence of an applied field. Another objective of the present invention is to suppress such spontaneous bubble formation.